Mono-ADP-ribosylation is a posttranslational modification of proteins which is involved in the action of bacterial toxins and in the regulation of cellular processes. Arginine-, cysteine- and aparagine-specific ADP- ribosyltransferases have been identified in animal tissues. NAD:arginine ADP-ribosyltransferase and ADP-ribosylarginine hydrolases, which catalyze the forward and reverse arms of a putative ADP-ribosylation cycle, have been identified in mammalian tissues. ADP-ribosylarginine hydrolase was purified from turkey erythrocytes and rat brain and its CDNA was cloned from the latter tissue. In the case of the arginine-specific ADP- ribosyltransferase, partial purification from rabbit skeletal muscle membranes was reported. To define the structure of this enzyme, it was purified, microsequenced, and cloned. Rabbit skeletal muscle arginine-specific ADP-ribosyltransferase was purified about 215,000-fold by sequential five-step chromatography. On the basis of the amino acid sequences of HPLC-purified tryptic peptides, degenerate oligonucleotide primers were synthesized and used in a polymerase chain reaction (PCR)-based procedure to generate CDNA. A specific probe, based on PCR-generated sequence, was used to screen a rabbit skeletal muscle library. A composite sequence, obtained from library screening and rapid amplification of the 5' end of the CDNA, contained a 981-base-pair open reading frame, encoding a 36,134-Da protein. Escherichia coli cells transformed with an expression vector containing transferase-specific sequence expressed transferase activity. A transferase-specific oligonucleotide probe recognized a 4-kilobase MRNA expressed primarily in rabbit skeletal and cardiac muscle. There was no extended similarity in deduced amino acid sequences of the muscle transferase and several ADP-ribosylating bacterial toxins.